Bone tissues of mammal repeat generation and resorption of bone. The tissue operates as a central point of calcium metabolism to keep the balance between bone resorption and generation in the growth period and even after the period of maturity. Bone resorption and resorption are well balanced by crosstalk between osteoclasts and osteoblasts. However, unbalance between bone resorption and generation lead metabolic bone diseases, including osteoporosis, rheumatoid arthritis, osteoarthritis, decrease of bone quantity due to diabetes, many types of hormone abnormalities, nutritional disorder, osteopetrosis and osteomalacia. Cellular pathogenesis of the most of above disorders remain to be elucidated. To resolve the issue and discover therapeutic agents for the metabolic bone diseases, methods for isolation and characterization of osteoclasts and osteoblasts have been required.
It has been studied well for isolation of osteoclasts of mice or rats. Recentry, Fujisawa et al. reported that human osteoclasts were obtained from patients with rheumatoid arthritis (Annals of Rheumatic Diseases. 55;816-822, (1996)). In their study, human osteoclasts were obtained in the presence of a mouse osteoblasts-like cell line, since it has been believed that the presence of accessory cells, such as osteoblasts or bone marrow stromal cells, were indispensable for differentiation of preosteoclasts in vitro. The accessory cells have been believed to play important role for osteoclastogenesisi by close contact with preosteoclasts.
On the other hand, JP 7-194373 describes a method for the differentiation of bone mallow cells into osteoblasts, osteoclasts or chondrocytes in a medium without bone mallow stromal cells or osteoblasts. But, they did not succeed in the isolation of osteoclasts itself. Under the circumstances above, in order to elucidate the mechanism of differentiation of preosteoclasts into osteoclasts, it was required to establish a method for isolation of preosteoclasts, and that for inducing the differentiation into osteoclasts, which needs few factors for the differentiation, and particularly, especially in the absence of accessory cells.